HRP20010797A2 - eNOS MUTATIONS USEFUL FOR GENE THERAPY AND THERAPEUTIC SCREENING - Google Patents

Description

References related to related applications

These application claims are related to U.S. Application 60/129,550 filed Apr. 16, 1999, which is incorporated herein by reference.

Announcement of public support

This invention was created as part of research funded by the following public funds: HL 57665 and HL 6137.

Technical area

This invention relates to novel variants or mutants of NOS that have a structural change at the site of Akt-dependent phosphorylation. Applied protein or peptide NOS, and the nucleic acid molecules that encode them are useful as gene therapy for the treatment of diseases that include post-angioplasty restenosis, diabetes and diseases of defective angiogenesis.

Previous knowledge

Atherosclerosis and vascular thrombosis are the main causes of morbidity and mortality, leading to coronary artery disease, myocardial infarction and stroke. Atherosclerosis begins with a change in the endothelium that is in the blood vessels. Alteration of the endothelium can ultimately result in the development of endothelial lesions marked, in part, by the entry of oxidized low-density cholesterol (LDL). Rupture of these lesions can lead to thrombosis and occlusion of blood vessels. In the case of a coronary artery, the rupture of the lesion complex can cause a myocardial infarction, while in the case of a carotid artery, a stroke can occur.

In coronary atherosclerosis, endothelial disruption can reduce the production of vasodilators, such as nitric oxide. Myocardial ischemia occurs when autoregulated vasodilation is prevented, either by flow-limiting coronary artery stenosis or by endothelial disruption. In both cases, blood flow through the artery cannot be further increased by a proportional increase in oxygen demand. In other situations, myocardial ischemia can occur when oxygen demands are constant but there is a primary disease in the coronary blood flow mediated by coronary artery spasm, so the rapid formation of atherosclerotic plaque leading to a reduction in the caliber of the coronary artery lumen, and/or occasional microvascular occlusion due to platelet aggregates .

Balloon angioplasty is commonly used to reopen a blood vessel that has been narrowed by plaque. Although balloon angioplasty is successful in a large percentage of cases in opening the vessel, the procedure often removes the endothelium and injures the vessel. This damage causes vascular smooth muscle cells in the blood vessel to migrate and proliferate into the area of injury, forming lesions known as myointimal hyperplasia or restenosis. A new lesion leads to return of symptoms within six months after angioplasty in a large proportion of patients.

With atherosclerosis, thrombosis and restenosis, there is also a loss of normal vascular functions, so such vessels shrink rather than expand. Significant vasoconstriction causes further narrowing of the blood vessel lumen and limits blood flow. This causes symptoms such as angina (such as heart artery involvement) or transient cerebral ischemia (ie a "small stroke" when a vessel in the brain is affected). This abnormal vascular function (significant vasoconstriction or inadequate vasodilation) also occurs in other diseases. Hypertension (high blood pressure) is caused by significant vasoconstriction as well as thickening of the vessel wall, especially in smaller blood vessels. This process can affect the vessels of the lungs and cause pulmonary (pulmonary) hypertension. Other disorders associated with significant vasoconstriction or inadequate vasodilation include transplant atherosclerosis, congestive heart failure, toxemia or pregnancy, Raynaud's phenomenon, Prinzmetal's angina (coronary vasospasm), cerebral vasospasm, hemolytic uremia, and impotence.

A substance released by the endothelium, originally called "endothelium-derived relaxing factor" (EDRF), plays an important role in inhibiting these pathological processes. EDRF is now known to be nitric oxide (NO). NO plays many roles in human physiology, including smooth muscle relaxation, inhibition of platelet aggregation and inhibition of mitogenesis, smooth muscle proliferation, and leukocyte adhesion. Because NO is the most potent endogenous vasodilator and because it is largely responsible for exercise-induced vasodilation in arteries, increasing NO synthesis may also improve exercise capacity in normal individuals and those with vascular disease.

Endothelial nitric oxide synthase (eNOS) is an isoform of nitric oxide synthase (NOS) that is responsible for the maintenance of systemic blood pressure, for vascular remodeling and angiogenesis (Shesely, at al., 1996; Huang et al., 1995; Rudic et al. 1998; Murohara et al., 1998). As a deficit in endothelial NO production is an early and persistent characteristic of atherosclerosis and vascular injury, eNOS has been proven to be an attractive target for vascular gene therapy. While the regulation of eNOS activation remains largely undefined, eNOS is known to be phosphorylated in response to various forms of cellular stimulation (Michel et al., 1993, Garcia-Cardena et al., 1996, Corson et al., 1996), however, the role of phosphorylation in the regulation of nitric oxide (NO) production and the responsible kinase (kinase) has not been previously elucidated.

Summary of the invention

The results of this invention, which derive in part from the new discovery that the serine/threon protein kinase, Akt (protein kinase B) can directly phosphorylate eNOS or a serine residue corresponding to residue 1197 in bovine eNOS or residue 1177 in human eNOS and activate the enzyme leading to NO production. Mutated eNOS (S1179A or S1177A) is resistant to Akt phosphorylation and activation, while mutant eNOS (S1179D and S1177D) or (S1179E and S1177E) is active. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and increases stimulation of VEGF-stimulated NO production in Akt-deficient silenced cells. Therefore, eNOS can be described as a substrate-dependent transduction through Akt, which releases the second messenger, NO. This invention is also based in part on the discovery that mutated eNOS (S1179D) exhibits an increased rate of NO production and increased reductase activity.

The present invention includes polypeptide or protein NOS, and isolated nucleic acids encoding them, wherein the polypeptide or protein NOS contains a substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS, or residue 1415 of human nNOS. A preferred substitution involves an amino acid with a negatively charged R group, including aspartic and glutamic acids.

The present invention also includes polypeptide or protein NOS, and isolated nucleic acids encoding them, wherein the polypeptide or protein NOS contains a substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS, or residue 1415 of human nNOS. A preferred substitution includes an amino acid in which the R group is not negatively charged, such as alanine.

This invention shows methods of stimulating collateral vessel development in ischemic disease with deficient endogenous angiogenesis, specific peripheral vascular disease and/or myocardial ischemia in patients, and includes the delivery of transgenes encoding polypeptide NOS from the invention or Akt polypeptide.

The invention further provides non-human transgenic animals that express the polypeptide NOS of the invention.

Finally, the invention includes methods of identifying agents that modulate Akt-regulated NOS activity, comprising the following general steps: (a) exposing to the agent purified NOS, preferably eNOS or nNOS or a cell expressing NOS, preferably eNOS or nNOS, and Akt; and (b) measuring Akt-regulated NOS activity, preferably eNOS or nNOS.

Short description of the pictures

Figures 1A-1B. Wild-type Akt but not kinase-inactive Akt increases NO release from cells expressing membrane-associated eNOS. In Figure 1A, COS cells were transfected with plasmids carrying the gene for eNOS in the absence or presence of Akt or kinase-inactive Akt (K179M) and NO production (tested as NO2") was determined by chemiluminescence. In Figure 1B, COS cells were transfected with plasmids carrying the eNOS gene as above. In Figure 1A and Figure 1B, values for NO2- production are subtracted from the level obtained from cells transfected with β-galactosidase cDNA alone. The inset shows protein expression in total cell lysate. Data are mean ±standard deviation, n=3-7 experiments, * indicates p<0.05.

Figures 2A-2D. Phosphorylation of eNOS by active Akt both in vitro and in vivo. In Fig. 2A, COS cells were transfected with HA-Akt or Ha-Akt(K179M), lysates were immunoprecipitated and placed in an in vitro kinase reaction with histone 2B (25 mg) or recombinant eNOS (3 mg) as substrate. The upper part shows the incorporation of 32P into the substrate, and the lower part shows the amount of substrate by Coomassie staining of the gel. In Figure 2B, 32 P-labeled eNOS wild-type or serine mutant (eNOS S635/1179) was affinity purified from transfected COS cells and subjected to autoradiography (top image) or Western blotting (bottom image). The graphical data in Figure 2B show the relative amount of labeled protein to the amount of immunoreactive eNOS and the gel. In Figure 2C, labeled eNOS was digested with trypsin and peptides were resolved by RP-HPLC. The upper chromatogram shows the predominantly labeled peptide with trypsin co-moving with an unlabeled synthetic phosphopeptide standard (lower chromatogram). The insets show by linear mode MS that the labeled peptide (top) and the standard phosphopeptide are of identical ion mass. In Figure 2D, recombinant wild-type eNOS or eNOS S1179A were purified and equal amounts (2.4 mg) were placed in an in vitro kinase reaction with recombinant Akt as described in Methods. The upper part of Figure 2D describes the incorporation of 32P into eNOS, and the lower part shows the amount of substrate by Coomassie staining of the gel. Graphical data (n=3) show the relative amount of labeled eNOS to the mass of eNOS (Comassie) in the in vitro kinase reaction.

Figure 3. Evidence that serine 1179 is functionally important for Akt-stimulated NO release. COS cells were transfected with a plasmid for wild-type eNOS or eNOS mutants, in the absence or presence of Akt, and protein expression and NO production (defined as NO2-) were determined. Interestingly, constructs with the S1179 mutation in A were not inactivated by Akt, and the S1179 to D mutation resulted in gain of function. In A, data are mean±SD of 4–7 experiments; * indicates a significant difference (p<0.05).

Figures 4A-4C. Akt regulates basal and stimulated production of NO in endothelial cells. In Figure 4A, BLMVEC were infected with adenoviral constructs (p-gal as control, myr Akt and AA-Akt) and the amount of NO2- produced was determined over 24 hours, (n=3). The inset shows the expression of eNOS and Akt. In Figure 4B, lysate from adenovirus-infected BLMVEC was tested for NOS activity. The same amounts of protein (50 mg) were incubated with different concentrations of free calcium and NOS activity was determined (n=3 experiments). In Figure 4C, BLMVEC were infected with adenoviruses as above, then stimulated with VEGF (40 ng/mL) for 30 min and NO2- release was measured by chemiluminescence. Data show VEGF stimulated release of NO2- after rejection of basal level. Data are mean±SD of 4–7 experiments; * indicates a significant difference (p<0.05).

Figures 5A and 5B. Purity and dimer/monomer ratio of wild-type and eNOS S1179D. In A and B, SDS-PAGE analysis was performed on a 7.5% polyacrylamide gel stained with Coomassie Blue. The molecular mass of the standard (line 1) and its size in kDa are shown on the left. Wild-type eNOS (lane 2) and eNOS S1179D (lane 3) (1 μg each) were resolved on SDS-PAGE at 4 °C. The molecular weight of the standard is in lane 1. Unheated wild-type and eNOS S1179D samples are separated in lanes 2 and 3. In lane 4, wild-type eNOS is heated to boiling in SDS sample buffer.

Figures 6A and 6B. eNOS 1179D has a higher rate of NO production (A) and reductase activity (B) than wild-type eNOS. In A, the rate of NO generation from wild-type (•) and S1179D (o) was determined, using a hemoglobin capture assay, as a function of L-arginine concentration, and the data are shown as a double reciprocal curve. In B, an assay was performed with DCIP and cytochrome c in the presence or absence of CaM. Values are mean ± standard deviation of 4-6 determinations. Similar results were obtained with at least three enzyme preparations. Significant differences between wild-type and S1179 eNOS (p<0.05) are indicated by an asterisk.

Figures 7A and 7B. NOS (A) and NADPH-dependent reductase activities were increased with eNOS S1179D compared with the wild-type enzyme. Assays of hemoglobin capture (a) and NADPH-dependent reduction of cytochrome c (B) were performed with wild-type and S1179eNOS. In A, the ratio of NO production was determined in the presence of all NOS cofactors (wild type (filled symbols) and S1179D (open symbols). Cytochrome c reduction rate measurement was performed in the absence of arginine and BH4 (a) for wild type (circles) and S1179D (triangles ) in the presence (filled symbols) or absence (open symbols).Values are mean ± standard deviation, n=3-6 determinations of at least three enzyme preparations.

Figures 8A-8D. Calmodulin- and calcium-dependent activation of NOS reductase activity is slightly increased by S1179 eNOS. Calmodulin-dependent hemoglobin capture (A) and cytochrome c reduction (B) were performed with wild-type (filled symbols) and S1179D eNOS (open symbols). The rate of NO production was detected by henoglobin capture in the presence of all NOS cofactors, while cytoctome c reduction was performed in the absence of arginine and BH4. In C and D, an identical experiment was performed in the presence of increasing concentrations of free calcium. The insets in Figures C and D describe the calcium-dependent degradation/synthesis of wild-type S1179D eNOS in the NO production and cytochrome c assays. Maximum turnover rate was monitored for wild type and S11798 eNOS: A, 22 and 43 min-1; B, 620 and 1400 min-1. Values are mean ± standard deviation, n=3-6 determinations of at least three enzyme preparations.

Figures 9A and 9B. EGTA-initiated NOS inactivation is reduced in S1179D eNOS. The hemoglobin capture assay (A) and the reductase assay (B) were performed as previously described, with the following modifications. The reaction was monitored for 1 min to determine the initial rate, then EGTA was added to the reaction mixture and the rate was monitored for an additional 1 min. The concentration of free calcium in the reaction is 200 μM, and enough EGTA was added to make the final chelator concentration 0, 200, 400 and 600 μM. Specific activity is normalized to 100% for wild-type and S1179D eNOS. Values are mean ± standard deviation, n=3-6 determinations from at least three enzyme preparations, nd indicates undetectable for wild-type eNOS.

Detailed description of the invention

A. General description

This invention is based in part on the discovery that the serine/threonine protein kinase, Akt (protein kinase B) can directly phosphorylate eNOS at serine 1179 (serine 1177 in human eNOS) and activate the enzyme leading to NO production, while mutant eNOS (S1179A) is resistant on Akt phosphorylation and activation. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and increases stimulation of VEGF-stimulated NO production in Akt-deficient silenced cells. Therefore, eNOS can be described as a substrate-dependent transduction through Akt, which releases the second messenger, NO. This invention is also based in part on the discovery that mutated eNOS, for example S1179D, exhibits an increased rate of NO production and increased reductase activity.

The demonstration that NO production is regulated by Akt-dependent eNOS phosphorylation provides new constitutively active eNOS mutants for use in gene therapy aimed at improving endothelial function in cardiovascular diseases associated with dysfunction of NO synthesis and biological activity. Such diseases include post-angioplasty restenosis, hypertension, atherosclerosis, heart failure including myocardial infarction, diabetes and diseases with defective angiogenesis. This discovery also provides a new therapeutic target useful in the design of drugs useful for the treatment of diseases associated with dysfunction of NO synthesis and biological activity.

The present invention also provides novel constitutively active nNOS mutants having a substituted amino acid corresponding to 1412 rat nNOS or 1415 human nNOS for use in gene therapy aimed at treating disease.

B. Specific units

Production of protein or polypeptide mutants

The present invention features proteinaceous or polypeptide NOS, allelic variants of proteinaceous NOS, and conservative substitution of proteinaceous NOS, all containing a mutation of a serine residue that is at the site of Akt-mediated phosphorylation. For example, proteins or polypeptides of the invention include, but are not limited to: (1) human protein eNOS containing a serine mutation at residue 1177 (Janssens et al. (1992), J. Biol. Chem. 267:14519-14522 which is incorporated by reference in its entirety) to another amino acid, such as alanine, and thus are resistant to Akt-mediated phosphorylation; (2) bovine protein eNOS containing a serine mutation at residue 1179 (SEQ ID NO: 2 in U.S. Patent 5,498,539 which is incorporated herein by reference in its entirety), to another amino acid, such as alanine, and are therefore resistant to Akt-mediated phosphorylation; (3) human eNOS proteins that contain a mutation of serine at residue 1415 to another amino acid, such as alanine, and are therefore resistant to Akt-mediated phosphorylation; (4) rat eNOS proteins that contain a mutation of serine at residue 1412 to another amino acid, such as alanine, and are therefore resistant to Akt-mediated phosphorylation; (5) human protein nNOS containing a serine mutation at residue 1415 to an amino acid containing a negatively charged R group, such as aspartic or glutamic acid, and are constitutively active and exhibit increased NO production and increased reductase activity; (6) bovine protein eNOS containing a serine mutation at residue 1179 to an amino acid containing a negatively charged R group, such as aspartic or glutamic acid, are both constitutively active and exhibit increased NO production and increased reductase activity; (7) human protein nNOS containing a serine mutation at residue 1415 to an amino acid containing a negatively charged R group, such as aspartic or glutamic acid, and are constitutively active and exhibit increased NO production and increased reductase activity; (8) rat protein nNOS containing a serine mutation at residue 1412 to an amino acid containing a negatively charged R group, such as aspartic or glutamic acid, are both constitutively active and exhibit increased NO production and increased reductase activity; (9) NOS proteins from non-human, bovine or rat species that have been modified to contain an amino acid other than serine at a position corresponding to serine at position 1177 of human eNOS or at position 11797 of bovine eNOS, position 1412 of rat nNOS, position 1415 of human nNOS, and which are resistant to phosphorylation by Akt and are constitutively active or show increased NO production and increased reductase activity. NOS mutants can also be created by mutation of other amino acids in the phosphorylation motif RXRXXS/T.

The present invention features constitutively active polypeptide NOS, preferably eNOS or nNOS that increase NO production and reductase activity and contain a mutation of a serine residue at the Akt downstream phosphorylation site. It is also within the art to obtain conservative variants of such polypeptide NOS mutants obtained by substitution, deletion, and insertion that increase NO production and reductase activity. As used herein, a conservative variant refers to an amino acid sequence change that does not adversely affect the ability of constitutively active NOS, preferably eNOS or nNOS to produce NO or increase the reductase activity of constitutively active NOS, preferably eNOS or nNOS. A substitution, deletion, or insertion is said to adversely affect constitutively active polypeptide NOS when the altered sequence affects the ability of constitutive NOS such that it does not produce NO at an elevated level and does not have increased reductase activity compared to wild-type NOS. For example, the overall charge, structure, or hydrophobic/hydrophilic properties of the constitutive NOS can be altered without adversely affecting the activity of the constitutive NOS. Therefore, the amino acid sequence of the polypeptide NOS can be changed so as to cause, for example, greater hydrophobicity or hydrophilicity of the polypeptide, without adversely affecting NOS activity.

As used herein, a "constitutively active" mutant or variant NOS, modified or isolated from a natural source, refers to a protein NOS, preferably eNOS or nNOS that produces NO at a higher rate than native NOS that contains serine in an unphosphorylated form at an amino acid residue corresponding to 1177 in human NOS or the rest of 1179 in bovine NOS. Preferred constitutively active variants contain amino acids having a negatively charged R group, such as aspartic or glutamic acid, at the amino acid residue corresponding to the serine at position 1177 of human eNOS or position 1179 of bovine eNOS.

The present invention features protein or polypeptide NOS, alanine variants of protein NOS, and conservative amino acid substitutions of protein NOS containing a substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat nNOS, and residue 1415 of human nNOS, wherein the substituted amino acid the remainder contains an R group that is not negatively charged, such as alanine.

Protein NOS, preferably protein eNOS or nNOs in this invention can be in isolated form. As used herein, a protein is said to be isolated when physical, mechanical, or chemical methods are used to remove the protein from cellular constituents normally associated with the protein. Those skilled in the art can easily use standard purification methods to obtain the isolated protein.

Also included in the invention are peptide NOS that bridge the Akt phosphorylation site corresponding to residue 1179 of bovine eNOS, 1177 of human eNOS, residue 1412 of rat nNOS, or residue 1415 of human nNOS. The peptides may contain serine at the phosphorylation site, preferably containing a serine substitution at a position corresponding to residue 1179 of bovine eNOS, 1177 of human eNOS, residue 1412 of rat nNOS or residue 1415 of human nNOS. Such substitutions include, but are not limited to, amino acids with an R group that mimics serine in the phosphorylated state, such as aspartic acid or glutamic acid. Such substitutions may include amino acids with an R group that is not negative, such as alanine. Peptides that bridge this site can be about 3, 5, 7, 10, 15, 17, 20, 25, 30, 40, 50 or more amino acids in length.

Protein or polypeptide NOS of the invention can be prepared by any convenient means, including recombinant expression of a cDNA for NOS that has been modified to replace or alter the nucleotide triplet encoding a serine corresponding to the serine at position 1177 of human eNOS, at position 1179 of bovine eNOS, at residue 1412 rat nNOS or in the rest 1415 human nNOS. Any available technique can be used to mutate the nucleotide triplet encoding the serine residue, such as homologous recombination, site-directed mutagenesis, or PCR mutagenesis (see Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). The starting cDNA may include human and bovine eNOS cDNA as well as cDNA encoding eNOS protein from other animal species including, but not limited to, rabbit, rat, laboratory rodent, pig, sheep, horse, and non-human primate species.

As used herein, a nucleic acid molecule encoding a proteinaceous or polypeptide NOS, preferably a proteinaceous or polypeptide eNOS or nNOS is said herein to be "isolated" when the nucleic acid molecule is sufficiently separated from contaminating nucleic acids encoding other polypeptides from sources of nucleic acids.

The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to small portions of sequences that encode an entire protein. The size of the fragment will be determined by the intended use. For example, if a fragment is chosen to encode an active part of a protein, it will need to be large enough to encode a functional region(s) of the protein, including the Akt phosphorylation site. If the fragment is to be used as a probe or primer for PCR, then the length of the fragment is chosen so that a relatively low number of false positives are obtained during probe/probe hybridization to the region surrounding the NOS Akt phosphorylation site.

Fragments of the encoding nucleic acid molecule of the present invention (i.e., synthetic oligonucleotides) used as probes or specific primers for the polymerase chain reaction (PCR), or for the synthesis of the gene sequence encoding the proteins of the invention can be easily synthesized by chemical techniques, for example, phosphotriether by the method of Matteucci et al.. 1981 J Am. Cham. Soc. 103:3185-3191) or using automated synthesis methods. Furthermore, larger DNA segments can be easily prepared by well-known methods, such as the synthesis of groups of oligonucleotides that define different modular gene segments, and after ligation of the oligonucleotides to build a complete modified gene.

The encoding nucleic acid molecules of the present invention may further be modified to contain a detectable label for diagnostic purposes. A number of such labels are known in the art that can be readily used with the encoding molecules described herein. Label drives include, but are not limited to, biotin, radioisotope-labeled nucleotides, and the like. One skilled in the art can use any labeling known in the art to obtain a labeled nucleic acid coding molecule.

The present invention provides recombinant DNA molecules (rDNA) containing the sequence encoding NOS, as described above. As used herein, an rDNA molecule is a DNA molecule that has undergone molecular manipulation. Methods for generating rDNA molecules are well known in the art, for example see Sambrook et al, Molecular Cloning (1989). In preferred rDNA molecules, the coding DNA sequence is operably linked to an expression control sequence and/or a vector sequence.

The choice of vector and/or sequence for expression control to which one of the protein family of coding sequences is operably linked in the present invention depends directly, as is known in the art, on the desired functional properties, e.g. on protein expression and on the host cell to be transform. The considered vector in this invention is capable of at least directing replication or insertion into the host chromosome, and preferably also expressing the structural gene included in the rDNA molecule.

An expression control element used to regulate the expression of an operably linked protein-coding sequence is known in the art and includes, but is not limited to: inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is easily controlled so that it is responsive to a nutrient in the medium of the host cell.

In one embodiment, the vector containing the encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence capable of directing autonomous replication and maintaining the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, thereby transforming it. Such replicons are well known in the art. Furthermore, vectors that include a prokaryotic replicon may also include genes whose expression is a detectable marker, eg for drug resistance. Typical bacterial genes responsible for drug resistance are those that display resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon may further include a prokaryotic or bacteriophage promoter capable of directing expression (transcription and translation) of the coding gene sequence in a bacterial host cell, such as E. coli. A promoter is an expression control element created from a DNA sequence that allows RNA polymerase to bind and transcription to occur. Promoter sequences that are compatible with bacterial hosts typically have a plasmid vector that contains a suitable restriction site for insertion of the DNA segment of the present invention. Typical such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Rochmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N.J.

An expression vector compatible with eukaryotic cells, preferably one compatible with vertebrate cells, can also be used in the form of rDNA molecules containing the coding sequence. Expression vectors for eukaryotic cells are well known in the art and can be obtained from several commercial sources. Typically, such vectors contain suitable restriction sites for insertion of the desired DNA segment. Typical such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), and similar eukaryotic expression vectors.

Eukaryotic expression vectors used to construct the rDNA molecules of the present invention may further include selectable markers that are effective in a eukaryotic cell, preferably markers for drug resistance. A preferred marker for drug resistance is a gene whose expression results in neomycin resistance, ie the neomycin phosphotransferase (neo) gene. (Suothern et al. (1982) J. Mol. Anal. Genet 1:327-341). Alternatively, the selectable marker may be present in a separate plasmid and the two vectors are introduced by simultaneous transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

The present invention further features cells transformed or transfected with a nucleic acid molecule encoding the protein NOS, preferably the protein eNOS or nNOS of the present invention. The host cell can be prokaryotic or eukaryotic. Eukaryotic cells useful for expressing proteins of the invention are not limited as long as the cell line is compatible with propagation of the expression vector and expression of the gene product. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as mouse, rat, monkey or human cell lines. Preferred eukaryotic host cells include Chinese guinea pig ovary (CHO) available from ATCC as CCL61, NIH Swiss mouse NIH/3T# embryo cells available from ATCC as CRL 1658, guinea pig kidney (BKH) cells and similar eukaryotic tissue culture cells. line. Any prokaryotic host can be used to express the rDNA molecule encoding the protein of the invention. The preferred prokaryotic host is E. coli, especially from constitutively active NOS mutants.

Transformation or transfection of appropriate host cells with the rDNA molecule of the present invention is accomplished by well-known methods that typically depend on the type of vector used in the host system. For the transformation of prokaryotic host cells, electroporation methods and cationic lipid or salt treatment with salt action are typically used, see, for example, Cohen et al. (1972) Proc. Natl. Acad Sci. USA 6 9:2100; and Maniatis et al., Molecular Cloning of a Laboratory Mammal. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). For the transformation of vertebrate cells, electroporation methods and the action of cationic lipids or salts are typically used, see, for example, Graham et al. (1983) Virol. 5 2:456; Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76:1373-76. Successfully transformed and transfected cells, i.e., cells containing rDNA molecules of the present invention, can be identified by well-known techniques including selectable marker selection. For example, cells generated by introducing the rDNA of this invention can be cloned to produce one type of colony. Cells from these colonies can be harvested, ligated and their DNA content examined for rDNA using the method described by Southern (1975) J. Mol. Biol. 98:503 or Bernet et al (1985) Biotech. 3:208 or proteins from cells tested by immunological methods.

The present invention further provides methods of producing proteinaceous NOS, preferably proteinaceous eNOS or proteinaceous nNOS of the invention using the nucleic acid molecules described herein. In general, the production of a recombinant form of a protein typically involves the following steps. First, a nucleic acid molecule encoding the protein of the invention was obtained. If the coding sequence is interrupted by an intron, it is directly suitable for expression in any host. The nucleic acid molecule is then preferably placed in operative linkage with suitable control sequences, as described above, and forms an expression unit containing the protein in the open reading frame. The expression unit is used to transform or transfect a suitable host and the transformed or transfected host is cultured under conditions that permit production of the recombinant protein. Recombinant protein can be isolated from the medium or from cells, and purification may not be necessary in some cases, and when some impurities can be tolerated.

Each of the previous steps can be performed in different ways. For example, the desired coding sequence can be obtained from genomic fragments and used directly in appropriate hosts. Construction of expression vectors active in different hosts was performed using appropriate replicons and control sequences, as shown above. Control sequences, expression vectors, and methods of transformation and transfection are dependent on the type of host cell used for gene expression and are discussed in detail earlier. Suitable restriction compounds may, if not normally available, be added to the ends of the coding sequence to allow gene insertion into these vectors. The skilled person may, however, adapt the host/expression system known in the art to use the nucleic acid molecules of the invention for protein production.

Gene therapy

Any suitable gene delivery system in combination with a gene expression system using the most convenient delivery route is encompassed by this invention. For example, mutant NOS or gene variants, preferably mutants or gene variants of eNOS or nNOS of the invention or Akt genes can be delivered to the heart (or skeletal muscle) including cardiac myocytes (and skeletal myocytes) in vitro or in vivo to direct the production of the encoded protein. Particularly useful are human Akt genes and NOS mutants, preferably human eNOS containing amino acids with a negatively charged R group, such as aspartic or glutamic acid at the position corresponding to serine 1177 in human eNOS. Routes of administration of the NOS mutant or gene variant include, but are not limited to, intravascular, intramuscular, intraperitoneal, intradermal, and intraarterial injection.

The adenovirus gene transfer system offers several advantages:

adenovirus can (i) accommodate a relatively large number of DNA insertions; (ii) be elevated to a high titer; (iii) infect a wide range of mammalian cell types; and (iv) be used in a large number of vectors containing different promoters. As adenoviruses are stable in the bloodstream, they can also be given by intravenous injection. The preferred delivery vector is helper cell-independent replication-deficient human adenovirus 5, although other delivery methods may be used, including delivery of nucleic acids directly into the cell of interest (see Sawa et al. (1998) Gene Ther. 5(11): 1472-80; Labhasetwar et al. (1998) J. Pahram. Sci. 8(11):1347-50; Lin et al. (1997) Hypertension 30:307-313; Chen et al. (1997) Circ. Res. 80(3):327-335; Channon et al. (1996) Cardiovasc. Res. 32:962-972; HarvHeart Lett. (1999) 9(8):5-6, and Nabel et al. (1999) Nat Med. 5(2):141-2.

It has been shown in vivo in myocardial cells that with a single intracoronary injection, using the adenovirus 5 system, the frequency of transfection is greater than 60% (Giordano and Hammond (1994) din. Res. 42:123A). Nonreplicative recombinant adenoviral vectors are also useful in the transfection of coronary endothelium and cardiac myocytes resulting in highly efficient transfection after intracoronary injection. Nonreplicative recombinant adenoviral vectors are also useful for transfection of desired cells in the peripheral vasculature (see U.S. Patent 5,792,453, which is incorporated herein by reference in its entirety).

Adenoviral vectors used in the present invention can be constructed by the "rescue" recombinant technique described by Graham et al. (1988) Virology 163:614-617. Briefly, the transgene encoding eNOS was cloned into a "shuttle" vector containing a promoter, a polylinker and an adenoviral sequence in which the E1A/E1B genes had been deleted. An example of a "shuttle" vector may be plasmid pAC1 (Virology 163:614-617, 1988) (or analog) which encodes a portion of the left end of the human adenoviral 5 genome (Virology, 163:614-617, 1988) minus the early protein encoding E1A and E1B sequences essential for viral replication, and the plasmid ACCMVPLPA (J. Biol. Chem. 267:25129-25134, 1992) containing a polylinker, CMV promoter and SV40 polyadenylation signal surrounded by a partial adenoviral sequence from which the EA/E1B genes were deleted. The use of plasmids PAC1 or ACCMVPLA facilitates the cloning procedure. The shuttle vector was then cotransfected with a plasmid containing the entire human adenovirus 5 genome of length too large to be encapsulated in 293 cells. Simultaneous transfection can be performed by calcium phosphate precipitation or lipofection (Biotechniques 15:868-872, 1993). Plasmid JM17 encodes the entire human adenovirus 5 genome plus part of the pBR322 vector including the resistance enhancer gene (4.3 kb). Although JM17 encodes all the adenoviral proteins necessary for the preparation of a mature viral particle, it is too large for encapsulation (40 kb vs. 36 kb for the wild type). In a small subset of simultaneously transfected cells, "rescue" recombination between a transgene containing a "shuttle" vector such as plasmid pAC1 and a plasmid carrying the entire adenovirus 5 genome such as plasmid pJM17 results in a gene lacking the E1A/E1B sequences and containing a transgene of of interest, but secondarily loses additional sequence such as the pBR322 sequence, and during recombination, is therefore small enough to be encapsulated. The beta-galactosidase-encoding HCMVAP1/acZ adenovirus driven by CMV (Clin. Res. 42:123A, 1994) can be used to assess gene transfer efficiency using an X-gal treatment assay.

In another embodiment, the gene encoding NOS, preferably eNOS or nNOS, can be introduced in vivo by an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV). , adenovirus and adenovirus-related virus (AAV). Also preferred are defective viruses, which completely or almost completely lack the viral gene. The defective virus is not infectious after entering the cell. The use of defective viable vectors is allowed for administration in specific localized sites of the cell without concern that the vector will infect the cell. Therefore, a specific site, eg in the brain or spinal cord can be specifically targeted by the vector. In a specific embodiment, defective herpes virus 1 (HSV1) can be used (Kaplitt et al. (1991) Molec. Cell. Neurosci. 2:320-330). In a further embodiment, the viral vector is an attenuated adenoviral vector such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626-630 (1992)). In another embodiment, the vector is a defective viral vector related to adenovirus (Samulski et al. (1987) J. Virol. 61:3096-3101; Samulski et al. (1989) J. Virsol. 63:3822-3828).

The present invention also contemplates the use of cell targeting not only by delivery of the transgene to, for example, the coronary artery or femoral artery, but also the use of tissue-specific promoters. By linking, for example, a tissue-specific transcriptional regulatory sequence of left ventricular myosin light chain 2 (MLC[2V]) or myosin heavy chain (MHC) to a transgene, such as the NOS-encoding genes of the invention within an adenoviral construct, transgene expression is restricted to ventricular cardiac myocytes. The efficiency of gene expression and degree of specificity with promoters (MLC[2V]) and MHC with lacZ was determined using the recombinant adenovirus system of the present invention. Cardiac-specific expression was shown earlier by Lee et al. (J. Biol. Chem. 267:15875-15885 (1992)). The promoter (MLC[2V]) contains 250 bp and fits easily within adenovirus 5. The myosin heavy chain promoter, which is known to be a strong transcriptional promoter, is a reasonably good alternative cardiac-specific promoter and contains less than 300 bp. Also available are smooth muscle cell promoters such as the SM22 alpha promoter (Kemp et al. (1995) Biochem. J. 310 (Pt 3):1037-43) and the SM alpha actin promoter (Shimizu et al. (1995)J . Biol. Chem. J. 270(13):7631-43). Other promoters such as the troponin-C promoter, although very effective and small enough, lack adequate tissue specificity. It is believed that using the promoter (MLC[2V]) or MHC and introducing the transgene in vivo, the cardiac myocyte alone (meaning without simultaneous expression in endothelial cells, smooth muscle cells and fibroblasts within the heart) will allow adequate expression of the protein NOS.

Limited cardiac myocyte expression is also suitable for use in gene transfer for the treatment of clinical myocardial ischemia. Restricted expression in the heart avoids the possible deleterious effect of angiogenesis in non-cardiac tissues such as the retina. Furthermore, of the cardiac cells, myocytes are likely to allow the longest transgene expression because the cells do not undergo rapid degradation/synthesis, and therefore expression would not decrease with both cell and death, as would happen with endothelial cells. Examples of endothelial specific promoters include the Tie-2 promoter (Schlaeger et al. (1997) Proc. Natl. Acad. Sci. 1;94(7):3058-63), the endothelial promoter (Lee et al. (1990) J. Biol . Chem. 265:10446-10450) and the eNOS promoter (Zhang et al. (1995) J. Biol. Chem. 270(25): 15320-6).

With respect to the treatment of heart disease, the present invention includes the targeting of the heart by intracoronary or intramuscular injection of a high titer vector and herein preferred transfection of all cell types. Diseases such as erectile dysfunction and cardiovascular disease including myocardial infarction, myocardial ischemia, heart failure, restenosis and stenosis, post-angioplasty stenosis and graft failure can be treated as described using a transgene encoding NOS, preferably a transgene encoding eNOS or nNOS .

Successful recombinant vectors can be purified from plaque by standard methods. The resulting viral vectors were propagated on 293 cells allowing E1A and E1B functions in trans at a preferred titer ranging from about 1010 to about 1012 virus particles/mL. Cells can be infected at 80% confluence and harvested 48 hours later. After 3 freeze-thaw cycles, cell debris was pelleted by centrifugation and virus was purified by CsCl gradient ultracentrifugation (double CsCl gradient ultracentrifugation is preferred). Before in ivvo injection, the viral sample was desalted by gel filtration over a Sepharose column such as G25 Sephadex. The product was then filtered through a 30 micron filter, thus reducing the harmful effect of intracoronary injection of unfiltered virus (cardiac arrhythmias described for life) and increasing efficient gene transfer. The resulting viral sample has a final viral titer in the range of 1010-1012 viral particles/mL Recombinant adenovirus must be of high purity, free of wild-type (potentially replicative) virus. Impure constructs can cause an intense immune response in the host animal. From this point of view, propagation and purification can be performed to exclude contaminants such as wild-type virus, for example, by identifying successful recombinants using the appropriate germline for PCR, performing two rounds of plaque purification and double CsCl gradient ultracentrifugation. Furthermore, problems associated with cardiac arrhythmias induced by injection of an adenoviral vector into a patient can be avoided by filtering the recombinant adenovirus through an appropriately sized filter paper prior to intracoronary injection. This strategy greatly improves gene transfer and expression.

The viral sample may be in the form of an injectable preparation containing a pharmaceutically acceptable carrier such as saline. The final titer of the vector and injectable preparation is preferably in the range of about 107 to about 1013 virus particles, which allows for efficient gene transfer. Other pharmaceutical carriers, formulations and dosages are described below. Adenoviral transgene constructs are introduced into the myocardium by direct intracoronary (or transplanted vessel) injection using standard methods based on the use of a percutaneous catheter under fluoroscopic guidance, at a quantity sufficient to express the transgene to such an extent that it leads to highly effective therapy. The injection can be introduced deep into the lumen (about 1 cm inside the arterial lumen) of the coronary artery (or transplanted vessel), and it is preferably introduced into both coronary arteries because the growth of the coronary blood vessels is very different within an individual patient. By injecting material directly into the lumen of the coronary artery with coronary catheters, it is possible to target the gene very efficiently and minimize the loss of recombinant vectors in the proximal aorta during injection. Gene expression, when directed in this way, is known not to occur in hepatocytes and no viral RNA can be found in urine at any time after intracoronary injection. Any type of coronary catheter or Stack perfusion catheter can be used in the present invention, for example. Furthermore, other techniques known to those skilled in the art can be used to transfer genes encoding NOS, preferably eNOS or nNOS, into the arterial wall.

For the treatment of peripheral vascular disease, a disease characterized by insufficient blood supply to the legs, a recombinant adenovirus expressing NOS, preferably the peptide or protein eNOS or nNOS of the invention, can be introduced by an inserted catheter into the proximal part of the femoral artery and arteries, and therefore act on gene transfer in skeletal muscle cells that receive blood and femoral arteries.

In cases where the transgene or nucleic acid encoding NOS, preferably eNOS or nNOS or protein Akt of the invention is first transfected into an endothelial or vascular smooth muscle cell in vitro, including the patient's own cells, the DNA can be transfected directly into the cells (see U. S. Patent 5,658,565). In general, to transfect target cells, a plasmid vector containing a DNA sequence encoding Akt or NOS of the invention or a corresponding biologically active fragment can be used in liposome-mediated transfection of the target cell. The stability of liposomes, coupled with the impermeable property of these vehicles, makes them useful for delivery of therapeutic DNA sequences (for a review article, see Mannino and Gould-Forgerite (1988) BioTechniques 6(7):682-690). Liposomes are known to be absorbed by many cell types via fusion. In one embodiment, cationic liposomes containing cationic cholesterol derivatives, such as SF-chol or DC-chol, are used. The DC-chol molecule includes a tertiary amino group, a medium-sized handle, and a carbamoyl linker, as described by Gao and Huang (Biochem. Biophys. Res. Comm. 179:280-285, 1991).

In a further embodiment, as far as lymphosomal technology is concerned, a viral or non-viral vector containing a DNA sequence encoding a biologically active NOS protein fragment, preferably an eNOS or nNOS protein fragment, is introduced into the target cell by transfecting the target cell with Lipofectamine (Bethesda Research Laboratory). Lipofectamine is a 3:1 liposomal formulation of the polyactive lipid 2,3-dioleyloxy-N-[2-(sperminecarboximido)ethyl]-N,N-dimethyl-1-propanaminium fluoroacetate (DOPSA) and the neutral lipid dioleoyl-phosphatidylethanolamine (DOPE).

Other non-viral methods of delivery include, but are not limited to: (a) direct injection of the DNA itself; (b) calcium phosphate [Ca3(PO4)2]-mediated cell transfection; (c) transfecting a mammalian cell by electroporation; (d) DEAE-dextran-mediated cell transfection; (e) polybrene-mediated delivery; (f) protoplast fusion; (microinjection); and (h) polylysine-mediated transformation, whereby the genetically engineered cells are returned to mammalian host cells.

Production of transgenic animals

Transgenic animals containing mutated genes encoding NOS, preferably genes encoding mutated eNOS or nNOS, as described herein, are also included in the invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often called a "transgene". The nucleic acid sequence of the transgene in the case of the NOS form can be integrated into the genome site where that specific nucleic acid sequence can normally be found or into the normal site of the transgene. A transgene may consist of a nucleic acid sequence derived from the genome of the same or different species than the target animal.

The term "germ cell line of transgenic animals" refers to transgenic animals in which a genetic change or genetic information has been introduced into the germ cell line, and therefore transgenic animals are enabled to transmit genetic information to their offspring. If such offspring do have some or all of their genetic information altered, they are transgenic animals.

The change or genetic information can be foreign to the species of animal to which the recipient belongs, only to a certain individual recipient, or it can be genetic information that the recipient already has. In the latter case, the applied or introduced gene may be expressed differently than the natural gene.

Transgenic animals can be produced by a variety of methods including transfection, electroporation, microinjection, introduction of genes into embryonic cells, and recombinant viral or retroviral infection (see U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al. (1993) Hypertension 22( 4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.) Recombinant Gene Expression Protocols, Methods and Molecular Biology No. 62, Humana Press (1997)) .

A number of recombinant or transgenic mice have been produced, including those expressing an activated oncogene sequence (U. S. Patent No. 4,736,866); express the simian SV 40 T-antigen (U. S. Patent No. 5,728,915); do not express interferon regulatory factor 1 (IRF-1) (U. S. Patent No. 5,731,490); show dopaminergic dysfunction (U. S. Patent No. 5,723,719); express at least one human gene involved in blood pressure control (U. S. Patent No. 5,731,489); shows greater similarity to the conditions that exist in the natural manifestation of Alzheimer's disease (U. S. Patent No. 5,720,936); have a reduced capacity to maintain cell adhesion (U. S. Patent No. 5,602,307); have the bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4): 1753-1760) or are capable of generating a full human antibody response (McCarthy (1997) The Lancer 349(9049):405).

While mice and rats remain the animals of choice for most transgenic experiments, in some cases the use of alternative animal species is preferred or even necessary. The transgenic procedure has been used successfully in a variety of non-rodent animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, guinea pigs, rabbits, cows, and guinea pigs (see, e.g., kim et al. (1997) Mol. Reprod. Dev. 46(4) :515-526; Houdebine (1995) Reprod. Nutr. Dev. 35(6) :609-617; Petters (1994) Reprod. FertH. Dev. 6(5):643-645; Schnieke et al.(1997) Science 278(5346):2130-2133, and Amoah (1997) J. Anima Science (75(2):578-585).

Any method of introducing nucleic acid fragments into recombinantly competent animal cells that favors simultaneous transformation of multiple nucleic acid molecules can be used. Detailed procedures for the production of transgenic animals are readily available to those skilled in the art, including those disclosed in U.S. Pat. Patent no. 5,489,743 and U.S. Pat. Patent no. 5,602,307. Furthermore, the production of NOS transgenic animals was developed. For example, transgenic mice have been produced that inducibly express or overexpress wild-type eNOS (see Ohashi et al. (1998), J. Clin. Invest 102(12):2061-71; and Drummond et al. (1998) J. Clin. , Invest. 102(12):2033-4). These methods can be used to produce mice expressing the NOS mutants of the invention.

Therapeutic sorting test

The discovery that phosphorylation of eNOS regulates its activity leaves room for the development of a screening assay to identify agents that modulate Akt-regulated NOS, preferably eNOS or nNOS. Any format can be used, including in vivo transgenic animal assays, protein-based in vitro assays, cell culture assays, and processing large numbers of samples.

In many drug screening programs that test the release of compounds, a large sample processing test is desirable to test the maximum number of compounds in a given time period. Assays performed in a cell-free system, such as many performed with semipurified proteins, are usually preferred as "primary" screening in that they can be generated to allow rapid development and relatively easy detection of a change in the target molecule mediated by the test compound. Moreover, the effect of cellular toxicity and/or bioavailability of the tested compound can generally be ignored in the in vitro system, and instead the test is focused primarily on the effect of the drug on the molecular target, which can be manifested by inhibition, for example of binding between molecules.

A cell or tissue culture-based assay can be performed, for example, by plating COS-7 cells (100 mm dishes) on nutrient medium, and transfecting plasmids with NOS (7.5-30 mg) and Akt (1 mg) using calcium phosphate. To balance all transfections, a cDNA expression vector for β-galatosidase can be cotransfected. Twenty-four or forty-eight hours after transfection, expression of the corresponding protein (40-80 mg) can be confirmed by Western blot analysis using eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOS pAb (Zymed Laboratories), or nNOSmAb (Zymed Laboratories).

Twenty-four or forty-eight hours after transfection, measurement of nitrite (NO2-) in the medium, a stable product of NO in aqueous solution, and NO specific chemiluminescence can be performed as described (Sessa et al., 1995). Media was deproteinized and samples containing NO2- were refluxed in glacial acetic acid containing sodium iodide. Under these conditions, NO2- is quantitatively reduced to NO, which is determined by a chemiluminescence detector after reaction with ozone in a NO analyzer (Sievers, Boulders, CO). In all experiments, controls can be prepared by inhibiting the release of NO2-, using NOS inhibitors. Furthermore, NO2- release from cells transfected with β-galatosidase cDNA can be subtracted from a control for baseline NO2- levels found in serum or medium. Accumulation of cGMP in COS can be used as a bioassay for NO production, as described. In an alternative format, conversion of 3H-L-arginine to 3H-L-citrulline can be used to determine NOS activity in COS cells or endothelial cell lysates, as previously described (Garcia-Cardena et al., 1998).

For in vivo phosphorylation assays, COS cells can be transfected overnight with cDNA for wild-type or S635 (control), bovine 1179A, D or E eNOS, human 1177D or E eNOS, rat 1412D or E nNOS, human 1415 D or E nNOS , and HA-Act. 36 hours after transfection, cells were placed in dialyzed saturated serum, phosphate-free Dulbecco's minimal medium supplemented with 80 μCi 32P orthophosphoric acid for 3 hours. Wortmannin (500 nM) in phosphate-free medium can be added to the cell aliquot beforehand for 1 h during labeling. The lysate was then collected, NOS was solubilized and partially purified by ADP Sepharose affinity chromatography as previously described, and the incorporation of 32P into NOS was visualized after SDS-PAGE (7.5%) by autoradiography and the amount of protein NOS was verified by Western blotting for NOS.

For in vitro phosphorylation studies, recombinant NOS purified from E. coli, eNOS purified from another source, or peptide NOS spanning the Akt phosphorylation site, were incubated with wild-type or with kinase-inactive immunoprecipitated Akt from transfected COS cells. Briefly, protein or peptide NOS were incubated with 32P g-ATP (2 mL, specific activity 3000 Ci/mmol), ATP (50 mM), DTT (1 mM) in buffer containing HEPES (20 mM, pH 7.4) , MnCl2 (10 mM) and immunoprecipitated Akt for 20 min at room temperature.

In experiments examining in vitro phosphorylation of wild-type or mutant NOS, recombinant Akt (1 mg) purified from baculovirus-infected SF9 cells was incubated with wild-type, S1179A bovine eNOS, S1177A human eNOS, S1179D or E bovine eNOS, S1177D or E human eNOS, S1412D or E rat nNOS or S1215D or E human nNOS using essentially the same conditions as above. Proteins can be separated by SDS-PAGE and the incorporation of 32P and the amount of protein determined by Coomassie staining as above.

The above screening assay that tests Akt-dependent phosphorylation or activation of NOS, preferably eNOS or nNOS, can be used for a wide variety of agents. For example, agents that inhibit NOS dephosphorylation (phosphatase inhibitors) and amino acids corresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat eNOS, or residue 1415 in human nNOS may be useful molecules in therapy. Similarly, agents that activate Akt or that mimic the Akt phosphorylation site on eNOS may be useful therapeutic molecules.

The means tested by the above methods may be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when it is selected without consideration of the specific sequence of a protein of the invention alone or associated with substrates, binding partners, etc. An example of randomly selected agents are agents selected using a chemical library or combinatorial peptide library or broth for the growth of organisms.

As used herein, an agent is said to be rationally selected or designed when the agent is selected to take into account the sequence of the target site and/or its conformation in relation to the action of the agent. For example, a rationally chosen peptide agent can be a peptide whose amino acid sequence is similar to the Akt phosphorylation site in NOS, especially peptides or small molecules that mimic the phosphorylated state in NOS.

The agents of this invention can be, for example, peptides, small molecules, vitamin derivatives as well as carbohydrates. One skilled in the art can readily recognize that there are no structural limitations to the means of this invention.

Peptide agents of the invention can be prepared standardly in the solid phase (or in the solution phase), by the method of peptide synthesis, as is known in the art. Furthermore, the DNA encoding these peptides can be synthesized using commercially available instrumentation oligonucleotide synthesis and prepared recombinantly using standard recombinant production systems. The use of solid-phase peptide synthesis is necessary if non-gene-encoded amino acids are involved.

The next class of agents in this invention are antibodies immunoreactive with the critical position of the protein of the invention. Antibodies are obtained by immunizing a suitable mammal with peptides containing the antigenic region, and these parts of the protein are targeted by the antibodies.

Use of agents found to modulate eNOS activity

Agents of the present invention, such as agents that inhibit the dephosphorylation of NOS (phosphatase inhibitors) at the amino acid corresponding to 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS, or residue 1415 of human nNOS, as well as agents that activate Akt or mimic Akt phosphorylation site on NOS can be administered by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and buccal routes. Alternatively or competitively, administration may be by oral route. The dose given will also depend on the age, health and weight of the recipient, the type of competing treatment if any, the frequency of treatment and the nature of the desired effect. As described below, there are many methods that can be easily adapted to provide these types of funds.

This invention further provides compositions containing one or more agents of the invention. As individual needs vary, determining the optimal range of effective amounts of each component is within the profession. A typical dose contains 0.1 to 10 mg/kg of body weight. A preferred dose contains 0.1 to 10 mg/kg of body weight. The most preferred dose contains 0.1 to 1 mg/kg of body weight.

In addition to the pharmaceutically active agent, the preparations of the present invention may contain pharmaceutically acceptable carriers containing excipients and auxiliaries that facilitate the processing of the active substance into a preparation that can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example in the form of water-soluble salts. Furthermore, suspensions of active compounds can be given as suitable oil suspensions for injections, for example with sesame oil or a synthetic ester of fatty acids, for example ethyl oleate or triglyceride. Aqueous suspensions for injection containing substances that increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may contain a stabilizer. Liposomes can also be used as means for encapsulating substances for introduction into the cell.

Pharmaceutical formulations for systemic administration according to the invention can be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation can be administered simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets including coated tablets, elixirs, suspensions, syrups or inhalers, and suitable controlled release agents.

In practicing the methods of the invention, the compounds of the invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In some preferred embodiments, the compounds of the invention can be administered simultaneously with other compounds that are typically prescribed for these conditions, and according to generally accepted medical practice, such as anticoagulants, thrombolytic agents or other antiplatelets, including platelet aggregation inhibitors, plasminogen activators in gourd, urokinase, prourokinase, streptokinase, heparin, aspirin or warfarin. The compounds of this invention can be used in vivo, usually in mammals, such as humans, sheep, horses, pigs, dogs, cats, rats, mice, or in vitro.

The following working examples specifically show preferred embodiments of the present invention, and are not intended to be limiting in any way to the remainder of the invention. Other generic configurations will be apparent to those skilled in the art.

EXAMPLES

The following procedures were used in Examples 1-2.

Cell transfection: cDNAs encoding bovine eNOS, human iNOS, rat nNOS in pcDNA3, and HA-tagged wild-type Akt, Akt (K179M), or myr-Akt in CMV6 were generated by standard cloning methods. Myr-nNOS in pcDNA3 was generated by PCR, incorporating new amino acid termini containing eNOS with an N-myristoyl group at the cosensus site (MGNLKSVG; SEQ 1 D NO: 1) fused in-frame to the second amino acid of the nNOS coding sequence. In preliminary tests of COS cells, an N-myristoyl group was introduced to that construct by incorporation of 3H-myristic acid, while natural nNOS was not, and this resulted in approximately 60% of the total target protein in the membrane fraction of the cell, while only 5-10% of nNOS was associated with the membrane in COS cells. Mutation of the potential Akt phosphorylation site in eNOS was generated with the Quick Site site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. All mutants were verified by DNA sequencing. COS-7 cells were plated on nutrient media (199 mm dishes) and transfected with plasmids for NOS (7.5-30 mg) and Akt (1 mg) using calcium phosphate. To balance all transfections, a cDNA expression vector for β-galatosidase can be cotransfected. Twenty-four or forty-eight hours after transfection, expression of the corresponding protein (40-80 mg) can be confirmed by Western blot analysis using eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOS pAb (Zymed Laboratories), or nNOSmAb (Zymed Laboratories).

Release of NO from transfected cells: 24-48 hours after transfection, the measurement of nitrite (NO2-), which is the stem product of NO in aqueous solution, can be performed in the medium, and NO specific chemiluminescence as described (Sessa et al., 1995 ). Proteins were removed from the medium and samples containing NO2- were fluxed in glacial acetic acid containing sodium iodide. Under these conditions, NO2- is quantitatively reduced to NO, which is determined by a chemiluminescence detector after reaction with ozone in a NO analyzer (Sievers, Boulders, CO). In all experiments, controls can be prepared by inhibiting the release of NO2- using NOS inhibitors. Furthermore, NO2- release from cells transfected with β-galatosidase cDNA can be subtracted from a control for baseline NO2- levels found in serum or medium. Accumulation of cGMP in COS can be used as a bioassay for NO production, as described.

NOS activity assay: Conversion of 3H-L-arginine to 3H-L-citrulline was used to determine NOS activity in COS cells or endothelial cell lysates, as previously described by Garcia-Cardena et al., (1998).

In vivo and iv vitro phosphorylation assays: For in vivo phosphorylation assays, COS cells were transfected overnight with cDNA for wild type or S635, and HA-Akt overnight. 36 hours after transfection, cells were placed in dialyzed saturated serum, phosphate-free Dulbecco's minimal medium supplemented with 80 μCi 32P orthophosphoric acid for 3 hours. Some cells were pre-supplemented with wortmannin (500 nM) in phosphate-free medium for 1 h during labeling. The lysate was collected, eNOS was solubilized and partially purified by ADP sepharose affinity chromatography as previously described and the incorporation of 32P into NOS was visualized after SDS-PAGE (7.5%) by autoradiography and the amount of protein NOS was verified by the Western blot method for NOS. For in vitro phosphorylation studies, recombinant NOS was purified from E. coli, incubated with wild-type or kinase-inactive Akt, and immunoprecipitated from transfected COS cells. eNOS was incubated with 32P g-ATP (2 mL, specific activity 3000 Ci/mmol), ATP (50 mM), DTT (1 mM) in a buffer containing HEPES (20 mM, pH 7.4), MnCl2 (10 mM) and with immunoprecipitated Akt for 20 min at room temperature.

In experiments examining in vitro phosphorylation of wild-type or mutant NOS, recombinant Akt (1 mg) purified from baculovirus-infected SF9 cells was incubated with wild-type or S1179A eNOS (2.4 mg, purified from E. coli) using essentially the same conditions as up. Proteins can be separated by SDS-PAGE and 32P incorporation, and the amount of protein determined by Coomassie staining as above.

In studies identifying labeled peptide eNOS, immunoprecipitated Akt was incubated with recombinant eNOS as above. The sample was applied to SDS-PAGE and the eNOS bands were resolved in the gel, and the resulting tryptic fragments were purified by RP-HPLC. Peptide mass and 32P incorporation was monitored and the prominently labeled signal was then analyzed by mass spectroscopy. In other experiments, peptides corresponding to a potential Akt phosphorylation site were synthesized, purified by HPLC and mass verified by spectrometry (W. M. Keck Biotechnology Resource Center, Vale University School of Medicine). The wild-type peptide was 1174RIRTQSFSLQERHLRGAVPWA1194 (SEQ. NO: 2) and the mutated peptide was identical except that S1179 was changed to alanine. In in vitro kinase reactions, such as those described above, peptides were incubated (25 mg) with recombinant Akt (1 mg). The reactions were then applied to phosphocellulose filters and the amount of incorporated phosphate was measured by Cerenkov counting.

Adenovirus infection and endothelial cell NO release: Bovine microvascular lung endothelial cells (BLMVEC) were cultured in 100 mm dishes (for basal NO release and NOS activity assay) or C6 wells (for NO stimulation) as previously described (Garcia-Cardena et al. 1996a). BLNVEDs were infected with 200 MOI adenovirus containing β-galactosidase 29 HA-tagged, inactive phosphorylated mutant Akt (A-A-Akt; Alessi et al. 1996) or terminal carboxy HA-tagged constitutively active Akt (myr-Akt) for 4 h. The virus was removed and the cells were left for 18 hours in complete medium. In preliminary studies with (3-galactosidase virus) these conditions were optimal for infection of 100% of cultures. To measure basal NO production, the medium was probed for NO release 24 hours after initial virus infection. To measure stimulated NO release, cells were then washed with with serum-free medium and then stimulated with VEGF (40 ng/mL) for 30 min. In some experiments, calcium dependence of NOS was determined 24 h after adenovirus infection. Infected cells were lysed in NOS assay buffer containing 1% NP40 and for activity, a detergent-soluble material was used.Lysates were incubated with EGTA buffered with calcium, to obtain adequate amounts of free calcium during incubation.

Statistics: Data are expressed as mean and standard deviation. Comparisons made by two-way Stident's test or ANOVA with post-hoc test were appropriate. Differences considered significant are p<0.05.

Example 1: Akt modulates the production of NO and eNOS

To examine the possibility that the downstream effector of PI-3 kinase Akt may directly affect NO production, COS-7 cells (which do not express NOS) were cotransfected with eNOS and wild-type Akt (HA-Akt) or with kinase-inactive Akt (HA- Akt K179M) and nitrite (NO2-) accumulation was measured by NO-specific chemiluminescence. Transfection of eNOS results in an increase in NO2- accumulation, an effect that is significantly enhanced by co-transfection of wild-type Akt, but not the kinase-inactive variant (Figure 1A). Identical results were obtained using cGMP as a bioassay for biologically active NO. Under these experimental conditions, Akt is catalytically active as determined by Western blotting with a phospho-Akt specific Ab (recognizing serine 473; not shown) and an Akt activity assay (see Figure 2A). Transfection of the constitutively active form of Akt (myr-Akt) increases cGMP accumulation (tested in COS cells) from 5.5±0.8 to 11.6±0.9 pmol cGMP/mg protein (cells transfected with eNOS alone or eNOS with myr-Akt) while it is inactive on the kinase Akt does not affect cGMP accumulation (5.8±0.8 pmol cGMP, n=4 experiments). As seen in the inset, equal levels of eNOS and Akt were expressed in COS cell lysate, demonstrating that Akt modulates eNOS and increases NO production under basal conditions.

eNOS is a doubly acylated protein from the peripheral membrane that localizes to the Golgi region and plasma membrane of endothelial cells (Liu et al., 1997; Garcia-Cardena et al., 1996a; Shaal et al., 1996) and compartmentalization is required for efficient NO production. in response to agonist challenge (Sessa et al. 1995; Lui et al., 1996; Kantor et al. 1996). To investigate whether activation of eNOS with Akt requires membrane compartmentalization, COS-7 were simultaneously transfected with cDNA for Akt and the amount of introduced myristyl group, pamitolyl group in defective mutant eNOS (G2A eNOS) and NO uptake were measured. As seen in Figure 1B, Akt did not activate the non-acylated form of eNOS demonstrating that membrane compartmentalization of both proteins is required for their functional interaction (Downward et al. 1998). It was then determined whether Akt can activate structurally similar but different NOS isoforms, neuronal and inducible NOS (nNOS and iNOS). Simultaneous transfection of Akt with nNOS and iNOS did not further increase NO release, demonstrating the specificity of Akt for eNOS. However, the addition of an N-myristoylation site to nNOS, to increase its interaction with biological membranes, leads to stimulation of nNOS with Akt in an analogous manner to that seen with eNOS, demonstrating that both isoforms may be responsible for activation of Akt kinase when membrane-anchored.

Example 2: Production of eNOS mutations

The above experiments show that Akt, perhaps via phosphorylation of eNOS, can modulate the release of NO from cells. Indeed, potential phorylation motifs (RXRXXS/T) are present in eNOS (serines 635 and 1179 in bovine eNOS or serines 633 and 1177 in human eNOS) and one motif is present in nNOS (serine 1412 in rat and 1415 in human nNOS). and was not found in iNOS. To examine whether eNOS is a possible substrate for Akt phosphorylation in vitro, COS cells were transfected with HA-Akt or HA-Akt (K179M) and kinase activity was assayed using a recombinant eNOS substrate. As seen in Figure 2A, active kinase phosphorylates histone 2B and eNOS (69±2.9 and 115.4±3.8 pmol ATP/nmol substrate, n=3), whereas inactive Akt does not significantly increase histone or eNOS phosphorylation. To elucidate whether a potential Akt phosphorylation site in eNOS is responsible for 32P incorporation, two serines were mutated with an alanine residue and the ability of Akt to stimulate wild-type and mutant eNOS phosphorylation was examined in intact COS cells. Transfected cells were labeled with 32P-orthophosphate, eNOS was partially purified by ADP-sepharose affinity chromatography, and the phosphorylation state and protein level were determined. As seen in Figure 2B, co-expression of Akt results in a twofold increase in eNOS phosphorylation relative to unstimulated cells. Pretreatment of eNOS/Akt transfected cells with wortmannin abolished the AKt-induced increase in phosphorylation. Furthermore, mutation of serine 634 and 1179 to an alanine residue abolishes Akt-dependent eNOS phosphorylation, demonstrating that these residues may be potential phosphorylation sites in whole cells.

To directly identify residues phosphorylated by Akt, wild-type eNOS was incubated with immunopurified Akt and phosphorylation sites were determined by HPLC followed by MALDi-mass spectrometry (MALDi-MS). As seen in Figure 2C, the primary 32 P-labeled tryptic phosphopeptide co-eluted with the synthetic phosphopeptide (amino acids 1177-1185 with phosphoserine at position 1179) and had an identical mass ion as determined by linear mode MS. Using reflectron mode MALDi-MS monitoring, the labeled tryptic peptide and standard phosphopeptide showed loss of H3PO4 indicating that the tryptic peptide was phosphorylated. In addition, mutation of S1179 in A significantly reduces Akt-dependent phosphorylation of eNOS compared to the wild-type protein (Figure 2D). Identical results were obtained using a peptide (amino acids 1174-1194) derived from wild-type or eNOS S1179A as a substrate for recombinant Akt (the wild-type peptide incorporated 24.6±3.7 mmol phosphate/mg compared to the alanine mutated peptide which incorporated 0.22±0.02 nmol phosphate/mg; n=5). These data indicate that eNOS is a substrate for Akt and that the primary phosphorylation site is serine 1179 (serine 1177 in human eNOS).

Next, we examined the functional significance of the potential Akt phosphorylation site serine 635 and identified the site serine 1179. Transfection of COS cells with double-mutated eNOS S635/1179A stops Akt-dependent NO release. Mutation of serine 635 to alanine does not reduce NO release, while eNOS S1179A reduces Akt-dependent activation of eNOS (Figure 3). These results indicate that serine 1179 is functionally important for NO release. Mutation of serine 1179 to aspartic acid (eNOS S1179D) to introduce a negative charge resulting in phosphate addition partially mimics the activated state induced by Akt (S1177D in human eNOS). All mutants are sufficiently expressed (see inset Western blots) and retain NOS catalytic activity in cell lysates (in COS cells transfected with eNOS alone, NOS activity was 85.3±27.0, 71.9±2.9, 80.8±23.2, and 131.8±36.7 pmol of generated L-citrulline/mg protein from lysates of COS cells expressing wild type, S1179A, S635, 1179A and eNOS 1179D, n=3 experiments).

To examine whether Akt mediates NO release from endothelial cells, bovine pulmonary microvascular endothelial cells (BLMVEC) were infected with adenoviruses expressing activated Akt (myr-Akt), activation-defective Akt (AA-Akt), or β-galactosidase as controls. accumulation of measured NO. As seen in Figure 4A, myr-Akt stimulates basal NO production from BLMVECs, whereas cells infected with β-galactosidase or insufficiently activated Akt release a low level of NO close to the limit of detection. These data together with similar results in COS cells indicate that Akt phosphorylation by eNOS is sufficient to regulate NO production at resting calcium levels. Indeed, NOS activity measured in lysate from myr-Akt-infected BLMVECs shows that the sensitivity of the enzyme to calcium activation, tested at a fixed concentration of calmodulin, is increased relative to NOS activity seen in BLMVECs initiated by β-galactosidase virus (Figure 4B). Interestingly, the sensitivity of NOS activity to calcium in infected cells with insufficiently active Akt is greatly attenuated in infected cells with myr-Akt and β-galactosidase.

Treatment of endothelial cells with VEGF (40 ng/mL) is known to activate Akt23 and NO release via a mechanism partially inhibited by PI-3 kinase inhibitors (Papapetrouos et al., 1997). To examine the functional link between VEGF and agonists for NO release and Akt activation, BLMVEC were infected with adenoviruses for myr-Akt, AA-Akt, or β-galactosidase, and VEGF-stimulated NO release was measured. As seen in Figure 4C, infection of endothelial cells with myr-Akt increased VEGF-induced NO production while AA-AKt decreased NO release. These results indicate that Akt participates in both the signal transduction events required for basal and stimulated NO production in endothelial cells.

All these data indicate that Akt can phosphorylate eNOS at serine 1179 (serine 1177 with human eNOS) and that phosphorylation increases the ability of the enzyme to generate NO.

Example 3

Materials and methods

eNOS constructs and protein purification - Wild-type bovine eNOS was expressed in plasmid pCW as previously described with groELS in E. coli BL21 cells (Martasek et al. 1996). The S1179D mutant eNOS for expression in E. coli was generated as follows. eNOS S1179D and pcDNA 3 (Fulton et al. 1999) was digested with XhoI/XbaI, subcloned into the identical sites of eNOS in pCW and co-expressed with groELS. Isolation of recombinant eNOS was performed as previously described (Roman et al., 1995; Martasek et al., 1999), with the following modifications. eNOS was eluted from 2'5'-ADP Sepharose with 10 mM NADPH or 10 mM 2'-AMP. The amount of eNOS was determined using absorbance at 409-412 nm with an extinction coefficient for heme content of 0.1 μM-1cm-1. Purity of eNOS was determined by 7.5% SDS-PAGE followed by Coomassie staining. Low-temperature SDS-PAGE was performed identically, except that samples were not heated to boiling and electrophoresis was performed at 4 °C in a mixture of ice and water (Klatt et al., 1995). In experiments in which NOS cofactors (L-arginine, calmodulin, and NADPH) were titrated, they were omitted from enzyme purification and storage and incubated as described below.

Assay for NOS activity: NO production was measured by hemoglobin capture as described (Kelm et al., 1988). Briefly, the reaction mixture contains eNOS (0.5-2.5 μg), oxyhemoglobin (8 μM), L-arginine (10 μM), BH4 (5 μM), CaCl2 (120 μM), calmodulin (120-200 nM) and NADPH (100 μM) in HEPES buffer (50 mM), pH 7.4. When determining the EC50 value of calcium for eNOS, the above mixture was modified as follows: MOPS buffer (10 mM, pH 7.6) was added. KCl (100 mM) and CaM (250 nM). Under these conditions, free calcium was calculated using WinMAXC program version 1.8 (Stanford University) with a Kd of 2.2x10-8. The exact concentration of free calcium was determined by mixing the appropriate ratio of 10 mM K2EGTA and 10 mM CaEGTA standard solution (Molecular Probes). Linear NOS activity was monitored for 2 minutes at 401 nm and NO production was calculated based on the change in absorbance using an extinction coefficient of 60 mM-1cm-1. All reactions were performed at 23 °C and each point represents 3-8 measurements. The extinction coefficient of 0.0033 μM-1cm-1 at 276 nm was used to determine the calmodulin concentration. NO production using this method was completely blocked by the addition of nitro-L-arginine (1 mM). When eNOS inactivation was determined by the addition of EGTA (200-800 μM), a chelator was added to the reaction mixture 1 min after the start of the reaction with NADPH. Identical conditions were used when NADPH-cytochrome c reductase activity was examined. These reactions contain CaM (120 nM) and CaCl2 (200 μM) in a volume of 0.5 mL with eNOOS (5 μg).

Conversion of L-arginine to L-citrulline was assayed as previously described by Bredt et al (1990). Briefly, eNOS (0.25-2 μg) was incubated for 3-10 minutes at 23 °C in the following reaction mixture: 3 pmol L-[3H]arginine (55 Ci/mmol), 10-300 μM arginine, 1 mM NADPH, 120-200 nM calmodulin, 2 mM CaCl2 and 30 μM BH4 in a final volume of 50-100 μL. The reaction was stopped by the addition of 0.5 mL of 20 mM HEPES, pH 5.5 containing 2 mM EGTA and EDTA. The reaction mixture was placed on a Dowex AG50WX8 and the flow was counted on a Packard 1500 liquid scintillation analyzer.

Reductase Activity Assay - NADPH-cytochrome c reductase activity and reduction of 2,6-dichlorophenolindophenol (DCIP) was measured as change in absorbance at 550 nm as described previously by Martasek et al (1999) and Masters et al (1967), using of the same extinction coefficient of 0.021 μM-1 for cytochrome c and DCIP. Briefly, the reaction mixture (1 mL) contains cytochrome c (90 μM); DCIP (36 μM), HEPES buffer (50 mM) at pH 7.6, NaCl (250 mM), NADPH (100 μM), calmodulin (120 nM), and CaCl2 (100 μM) or other substances, as indicated. The reaction was monitored for 60 s (at 23 °C) after the addition of eNOS. When the inactivation of the reducing activity was determined by the addition of EGTA (200-800 μM), the chelator was added 1 min after the initiation of the reaction and was monitored for another 1 min. The reaction contained HEPES buffer (50 mM) at pH 7.6, CaM (120 nM) and CaCl2 (200 μM) and was initiated with NADPH (100 μM). In experiments investigating eNOS inactivation with EGTA, NaCl was not added to mimic the conditions used in hemoglobin capture experiments. The addition of NOS inhibitors did not affect the reduction ratio of cytochrome c (not shown). Determination of EC50 calcium for eNOS was performed as described above for the hemoglobin capture assay.

Data analysis and statistics - All data are expressed as mean and standard deviation. At least three determinations were performed with at least three different batches of enzymes for each data set. Wild-type and mutant enzymes were purified simultaneously with the control due to variation in activity between preparations. Statistical significance was determined using the Student's t test, and p<0.05 is considered statistically significant.

THE RESULTS

Expression and Purification of eNOS - Wild-type and S1179D eNOS were expressed and purified from E coli. From a 1.6 liter culture, approximately 2.5-4.0 mg of eNOS was typically isolated using 2'5'-ADP Sepharose 4B chromatography. As seen in Figure 5A, both enzymes are >90% pure based on Coomassie staining. These results are typical, as seen from seven independent parallel preparations of eNOS wild type and S1179D. Both enzymes are primarily in their dimeric form, as shown by low-temperature SDS-PAGE (Figure 5B).

eNOS S1179D has higher NO synthase activity and reductase activity than wild-type eNOS - Next, the activities of wild-type and S1179D eNOS were compared by measuring the rate of NO production. S1179D eNOS shows a higher turnover rate (under optimal conditions) compared to the wild-type enzyme (84±6 vs. 27±1 min-1, n=6 separate paired enzyme preparations). Km values with L-arginine were similar for eNOS 1179D and wild-type (Figure 6A, 1.8 vs. 2.5 μM; see Table 1).

[image]

As the rate of electron flow from the reductase domain to the oxygenase domain is critical for NOS catalysis, the increase in S1179D eNOS activity was examined to determine whether it could contribute to the increase in reductase activity. When the reduction of DCIP and cytochrome c was examined, a significant increase in activity was observed for S1179D compared to wild-type eNOS (Figure 6B). Furthermore, the increase is accentuated by the presence of CaM, which increases the total activity of both enzymes. Basal reduction of cytochrome c in the absence of CaM was 4-fold higher with S1179D compared to wild-type eNOS. CaM-stimulated cytochrome c reduction was higher (749±35 vs. 1272±55 min-1 for wild-type and S1179D eNOS, n=3-5), however, the level of stimulation with CaM was 8-fold higher for wild-type eNOS and only 3 times higher for S1179D eNOS.

We next determined whether S1179D eNOS produces more superoxide than wild-type eNOS, which can reduce cytochrome c. As expected, no inhibitable reduction of cytochrome c by superoxide dismutase (as an index of superoxide anion generation) was observed in the absence of CaM, as previously shown for wild-type eNOS (278±9 vs. 288±7 min-1, n= 3-5). However, in the presence of CaM, superoxide dismutase reduces the rate of cytochrome c reduction for wild-type (610±51 vs. 866±8 min-1) and S1179D (1179±43 vs. 1518±19 min-1) eNOS. The relative reduction in cytochrome c activity in the presence of superoxide dismutase was similar for both enzymes (30% for wild-type and 22% for S1179D eNOS), indicating that the increased reductase activity of S1179D compared to wild-type eNOS (as tested by cytochrome c reduction) did not originate from enzyme unbinding.

NADPH-dependent NO formation and reductase activity are not different for wild-type eNOS and S1179D - NADPH dependence on NO production and cytochrome c reduction was investigated because the NADPH binding site lies close to Ser-1179 in eNOS. S1179D eNOS has a higher maximal turnover number (kcat) than wild type based on NO production, tested in the presence of CaM (Figure 7A). The increased kcat is associated with a fourfold increase in the Km for NADPH for S1179D eNOS compared to wild-type eNOS (36 vs. 8 μM). The Kcat for the NADPH-dependent reduction of cytochrome c in the absence of CaM is higher for eNOS S1179D than for wild-type eNOS (290 vs. 70 min-1, Figure 7B). In the presence of CaM, the kcat for cytochrome c reduction is significantly higher for S1179D than for wild-type eNOS (840 vs. 460 min-1). Km values for NADPH were unchanged in the absence and presence of CaM (0.40 and 0.75 μM for wild-type eNPOS and 2.0 and 1.9 μM for S1179D eNOS in the absence and presence of CaM).

EC50 values for CaM are unchanged for wild type and S1179D eNOS - To test whether the increased activity of S1179D eNOS originates from a change in the affinity of the enzyme for CaM, the kinetics of NOS activity and cytochrome c reduction were tested in the presence of all NOS cofactors in excess as a function of concentration CaM. Kcat for CaM activation of NOS activity is 22 min-1 for wild type and 43 min-1 for S1179D eNOS. By data transformation, normalization of different Kcat showed a small shift of the curve to the left for S1179D eNOS and a small difference in EC50 values for CaM, which is consistent with the data presented for phosphono-eNOS (Mitchell et al. 1999). EC50 values are 8 nM for wild type and 7 nM for S1179D eNOS (Figure 8A). NADPH-mediated reduction of cytochrome c was measured. The Kcat for CaM activation of cytochrome c reduction is about 2-fold higher for S1179D eNOS compared to the wild-type enzyme (1140 and 620 min-1 for S1179D and wild-type eNOS). Transformation of the data, which were normalized for differences in kcat, showed differences in EC50 values for CaM between wild-type and S1179D eNOS (21 vs. 13 nM, Figure 8B).

Comparison of eNOS activation and inactivation by calcium - Previous experiments have shown that the "apparent calcium sensitivity" of eNOS is increased in cells expressing bulk phosphono-eNOS or S1179D eNOS, indicating that phosphorylation altered the affinity of calcium/CaM activation (Dimmeler et al., 1999; Futon et al. 1999). As seen in Figure 8C, after normalization for differences in maximal activity, calcium dependence was slightly increased for S1179D eNOS (p<0.05, two-way analysis of variance). The EC50 values for calcium with wild-type and S1179D eNOS are also slightly different (310 and 250 nM calcium), as determined for NO production (in the presence of 250 nM CaM). As seen in the inset of Figure 8C, increasing free calcium concentrations indeed increased the S1179D eNOS abundance more than was seen with the wild-type enzyme. Furthermore, EC50 values for calcium assayed for cytochrome c reduction were similar to those obtained by measuring NO production (Figure 8D; 290 and 220 nM for wild-type and S1179D eNOS, respectively). Again, Vmax for calcium activation and turnover number for S1179D eNOS is greater than that for wild type (Figure 4D, inset).

To examine whether the inactivation of S1179D eNOS is different from that of the wild-type enzyme, the reduction of eNOS activity after calcium chelation with EGTA was measured. In these experiments, all NOS cofactors were added in the presence of calcium (200 μM) and NO production was monitored for 1 min, followed by the addition of various concentrations of EGTA and monitoring continued for an additional 1 min. As seen in Figure 9A, addition of EGTA to wild-type and S1179D eNOS reduced NO production in a concentration-dependent manner. However, NO production from S1179D eNOS is less sensitive to the addition of EGTA, i.e., wild-type eNOS activity declines more rapidly at lower EGTA concentrations than S1179D eNOS activity. The greatest difference in activity between the enzymes was seen at 400 μM EGTA. Furthermore, at 600 μM EGTA no activity was detected for wild-type eNOS, while residual activity was still detected for S1179D eNOS. Similar results were obtained using cytochrome c reduction (Figure 9B) with significant differences in activity seen with 400 and 600 μM chelator added to the reaction. However, at the highest concentrations of ETA, residual reductase activity was found for both wild-type and S1179D eNOS.

In conclusion, bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by protein kinase Akt at serine 1179 (Fulton et al. 1999) and human endothelial nitric oxide synthase (eNOS) is phosphorylated directly by protein kinase Akt at serine 1179 (Fulton et al. 1999). 1177 (Dimmer et al. 1999). Mutation of residue 1179 in bovine eNOS to negatively charged aspartate constitutively increases nitric oxide (NO) production in the absence of agonists.

It should be understood that the foregoing discussion and examples only represent a detailed description of some preferred entities. Therefore, it should be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All references, articles and patents listed above or below are reproduced here in their entirety.

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Claims (43)

1. An isolated nucleic acid molecule characterized by encoding a polypeptide NOS selected from the group consisting of constitutively active polypeptide NOS, polypeptide NOS having an increased rate of NO production and polypeptide NOS having an increased reductase activity. 2. The isolated nucleic acid molecule of claim 1, characterized in that the polypeptide NOS contains a substituted amino acid residue corresponding to NOS residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human nNOS. 3. The isolated nucleic acid molecule of claim 2, characterized in that the substituted amino acid residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human nNOS mimics phosphoserine. 4. The isolated nucleic acid molecule of claim 2, characterized in that the serine corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human nNOS is substituted with aspartic or glutamic acid. 5. The isolated nucleic acid molecule from claim 2, characterized in that the substituted amino acid contains an R group that mimics a phosphate group. 6. Polypeptide, characterized in that it is encoded by an isolated nucleic acid molecule from any of claims 1-5. 7. Isolated polypeptide NOS, indicated by the fact that it is selected from the group consisting of polypeptide NOS, polypeptide NOS with increased production of NO, and polypeptide NOS with increased reductase activity. 8. The polypeptide NOS of claim 7, characterized in that it contains a substituted amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human eNOS. 9. Constitutively active eNOS from claim 8, characterized in that the substituted amino acid is substituted with an aspartic or glutamic acid residue. 10. A method for stimulating the development of a coronary collateral blood vessel in a patient, characterized in that it contains the step of delivering a transgene encoding a polypeptide from claim 7 or an Akt polypeptide in a manner that is effective in promoting the development of a collateral blood vessel. 11. The method of claim 10, characterized in that the transgene is expressed from a promoter specific for a ventricular myocyte, a promoter specific for a smooth muscle cell or from a promoter specific for an endothelial cell. 12. The method of claim 11, characterized in that the ventricular myocyte-specific promoter has the promoter sequence of ventricular myosin light chain-2 or myosin heavy chain. 13. A method for stimulating the development of a blood vessel in a patient who has peripheral vascular deficiency disease, characterized in that it contains the step of delivering a transgene encoding a polypeptide from patent claim 7 or an Akt polypeptide in a manner that is effective in promoting the development of a peripheral vascular blood vessel. 14. A method of treating myocardial ischemia, characterized in that it contains the step of delivering a transgene encoding a polypeptide from claim 7 or an Akt polypeptide in a manner that is effective in the treatment of myocardial ischemia. 15. The method from claim 14, characterized in that the expression of the transgene is limited to cardiac myocytes. 16. The method from claim 15, characterized in that the expression of the transgene is limited to ventricular cardiac myocytes. 17. The method of any one of claims 10-16, characterized in that the transgene is encoded by a replication defective vector. 18. The method from claim 17, characterized in that the vector of defective replication is an adenovirus. 19. The method of any one of claims 10-16, characterized in that the transgene is transfected into a cell in vitro. 20. The method of claim 18, characterized in that the transfected cells are delivered to the patient. 21. A non-human transgenic animal, characterized in that it expresses the polypeptide of claim 7. 22. A method for identifying an agent that modulates Akt-regulated NOS activity, comprising the steps of: (a) exposure to the agent of the NOS-encoding cell; you (b) measurement of NOS activity regulated by Akt, and thus identification of an agent that modulates NOs activity regulated by Akt.. 23. The method of claim 22, characterized in that step (b) comprises determining the state of phosphorylation of NOS on an amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human nNOS. 24. The method of claim 23, characterized in that the NOS is activated by an agent. 25. The method of claim 24, characterized in that the agent mimics eNOS phosphorylation mediated by Akt. 26. The method of claim 25, characterized in that the agent inhibits dephosphorylation of the amino acid residue corresponding to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of rat eNOS or residue 1415 of human nNOS. 27. The method of claim 22, characterized in that step (b) contains the measurement of NO production. 28. The method from claim 27, characterized in that NO is detected by measuring the nitrite concentration or by converting 3H-L-arginine to 3H-L-citrulline. 29. An in vitro method for identifying an agent that modulates NOS activity regulated by Akt, characterized in that it contains the following steps: (a) exposure to an agent of Akt and protein or peptide NOS; you (b) measurement of NOS activity. 30. The method from claim 29, characterized in that NOS activity is measured by determining the Akt-dependent NOS phosphorylation state. 31. The method of claim 30, characterized in that the peptide NOS contains SEQ ID NO:2. 32. The method from claim 30, characterized in that in step (b) the NOS activity is reductase activity. 33. The method from patent claim 30, characterized in that in step (b) the NOS activity is the production of NO from NOS. 34. An isolated nucleic acid molecule, characterized in that it encodes a polypeptide NOS selected from the group consisting of bovine eNOS having a substituted acid residue corresponding to residue 1179, human eNOS having a substituted acid residue corresponding to residue 1177, rat eNOS having a substituted acid residue corresponding to residue 1177 corresponds to residue 1412, and human nNOS which has a substituted acid residue corresponding to residue 1415, and the substituted amino acid residue contains an R group that is not negatively charged. 35. The isolated nucleic acid molecule of claim 34, characterized in that the substituted amino acid residue is an alanine residue. 36. Isolated polypeptide NOS, indicated by this being selected from the group consisting of bovine eNOS having a substituted acid residue corresponding to residue 1179, human eNOS having a substituted acid residue corresponding to residue 1177, rat eNOS having a substituted acid residue corresponding to residue 1412, and human nNOS having a substituted acid residue corresponding to residue 1415, and the substituted amino acid residue contains an R group that is not negatively charged. 37. Polypeptide NOS from claim 36, characterized in that the substituted amino acid residue is an alanine residue. 38. The method of claim 11, characterized in that the specific endothelial cell promoter is Tie-2 promoter, endothelial promoter or eNOS promoter. 39. The method of claim 11, characterized in that the smooth muscle cell promoter is SM22 promoter or SM alpha actin promoter. 40. A method of treating cardiovascular disease in a patient, characterized in that it contains the step of delivering to the patient a transgene encoding a polypeptide from claim 7 or an Akt polypeptide. 41. The method from claim 40, characterized in that the cardiovascular disease is selected from the group consisting of: heart failure, myocardial infarction, restenosis, post-angioplasty stenosis, stent stenosis and loosening of the transplanted stent. 42. A method of treating erectile dysfunction in a patient, characterized in that it contains the step of delivering to the patient a transgene encoding a polypeptide from claim 7 or an Akt polypeptide. 43. The method of any one of claims 10, 13, 14, 40 or 42, characterized in that the transgene is delivered intravascularly, intramuscularly, intradermally, intraperitoneally or intraarterially.